Vaccine development for human parasitic diseases has not been a fruitful endeavor to date, in part due to the genetic complexity of these eukaryotic pathogens and the relative lack of understanding of effective host immune responses to, and immune evasion mechanisms used by them. In many sites, the transmission of Trypanosoma cruzi to insects - and subsequently to the people - is nearly totally determined by the presence or absence of T. cruzi infected dogs or cats in the home. Logistically what this means is that vaccination of dogs and cats so that they are not infectious to bugs could be a highly effective tool for the prevention human infections. We propose the development of avirulent lines of T. cruzi and their evaluation as vaccines to prevent the transmission of T. cruzi from companion or livestock animals to insects. Using the specific deletion of T. cruzi genes we will develop avirulent lines, selecting for their ability to grow well as epimastigotes and to convert to infective metacyclic trypomastigotes, to infect cells lines in vitro, to induce strong T cell and antibody responses in vivo in mice and ultimately, to prevent the development of detectable parasitemia in mice and dogs following challenge with virulent parasite lines. We will optimize the gene knockout (KO) avirulent lines to enhance their potency and safety by overexpression of low copy genes encoding non-variant T. cruzi proteins as well as by the expression and secretion of the heterologous protein TLR ligands from bacteria. These modifications are expected to accelerate and potentiate the immune response to T. cruzi. Among the enticing aspects of this approach is that a vaccine for companion animals would not have to absolutely prevent infection - just keep parasite levels in the blood of these animals below the level of transmissibility to insects. Additionally, such a vaccine could be delivered in an oral form - making widespread vaccination of animals quite easy. PUBLIC HEALTH RELEVANCE: Chagas disease (American trypanosomiasis) is the highest impact infectious disease in Latin America and a growing threat in the United States. The goal of this project is to develop and test a live, avirulent vaccine that will prevent vaccinated animals from serving as a source of infection for the reduviid bugs which vector this infection. Such a vaccine could become an integral tool in the armamentarium for the prevention of T. cruzi infection in humans.